CHARACTERIZATION OF NOVEL BUTADIENE-INDUCED PUTATIVE p53 TARGET GENES

描述（由申请人提供）：丁二烯（BD）是一种高产量化学品，目前被列为有害空气污染物。这种环境化学物质是一种已知的诱变剂和人类致癌物，具有广泛的造血、生殖和神经毒性，包括骨髓衰竭、卵巢、脾脏和胸腺萎缩。虽然已知双酚d的毒性作用是通过其活性代谢物介导的，但其毒性作用的细胞和分子机制尚不完全清楚。阐明双酚a毒性的分子机制是制定双酚a暴露治疗和干预方案的必要前提，也是本研究的长期目标。丁二烯最有效的活性代谢物，二氧丁烷（DEB），先前已报道可诱导p53调节的人淋巴细胞凋亡。尽管肿瘤抑制蛋白p53是一种多功能转录因子，在多种应激形式下介导细胞凋亡，但已知p53的作用和信号传导机制是细胞类型和损伤特异性的。对暴露于deba的淋巴细胞中p53作用机制的研究已经导致发现了新的deba诱导的假定p53靶基因。因此，本研究的目的是确定p53如何调节这些新的debb诱导的p53靶基因的表达，并确定这种调节在debb暴露的人类淋巴细胞中的功能方面。待验证的中心假设是，新的deb诱导的假定的p53靶基因在转录和/或转录后水平上受p53调控，从而在暴露的淋巴细胞中发出信号和/或执行deb诱导的p53依赖性凋亡。该项目将利用高通量方法，包括小干扰RNA、染色质免疫沉淀定量PCR测定、报告基因测定、聚合酶链反应阵列、原发性淋巴细胞以及基因配对p53缺陷和精通细胞系，通过两个特定目标来检验中心假设：(1)。确定p53如何在暴露于deb的人淋巴细胞中调控新的deb诱导的假定p53靶基因的表达。（2）. 确定新的DEB诱导的假定p53靶基因在暴露的人淋巴细胞内DEB的细胞和分子毒性中的作用。在这些研究完成后，我们的期望是发现新的deb诱导的直接和/或间接p53靶点，并推断它们在deb暴露细胞中的功能。总的来说，这些信息很重要，因为它不仅能让我们更好地理解丁二烯在细胞和分子水平上的毒性，还能让我们更好地理解p53在这一过程中的作用。这一知识将对丁二烯风险评估以及丁二烯暴露和致癌后更好的治疗干预手段产生重要的垂直影响。

DESCRIPTION (provided by applicant): Butadiene (BD) is a high production volume chemical that is currently regulated as a hazardous air pollutant. This environmental chemical is a known mutagen and human carcinogen, and possesses a wide range of hematopoietic, reproductive, and neurological toxicity, which includes bone marrow depletion, ovarian, spleen and thymus atrophy. Although the toxic effects of BD are known to be mediated through its active metabolites, the cellular and molecular mechanisms responsible for BD's toxic effects are not completely understood. Elucidation of the molecular mechanisms responsible for BD toxicity is a necessary pre-requisite to the development of therapeutic and intervention protocols for the management of BD exposure, and this is the long-term goal of this application. Butadiene's most potent active metabolite, diepoxybutane (DEB), has previously been reported to induce p53-regulated apoptosis in human lymphocytes. Although the tumor suppressor p53 protein is a multifunctional transcription factor that mediates apoptosis in response to various forms of stress, the mechanisms of p53 action and p53 signaling are known to be cell type and damage specific. Efforts towards the understanding of the mechanisms of p53 action in DEB-exposed lymphocytes have led to the discovery of novel DEB-induced putative p53-target genes. The objective of this proposal, therefore, is to determine how p53 regulates the expression of these novel DEB-induced putative p53-target genes, and determine the functional aspects of this regulation in DEB-exposed human lymphocytes. The central hypothesis to be tested is that the novel DEB-induced putative p53-target genes are regulated by p53 at the transcriptional and/or post-transcriptional level to signal and/or execute DEB-induced p53-dependent apoptosis in exposed lymphocytes. This project will utilize high-throughput approaches involving small interfering RNA, chromatin immunoprecipitation-quantitative PCR assays, reporter gene assays, polymerase chain reaction arrays, primary lymphocytes, as well as genetically-paired p53-deficient and proficient cell lines to test the central hypothesis through two specific aims: (1). Determine how p53 regulates the expression of the novel DEB-induced putative p53-target genes in DEB-exposed human lymphocytes. (2). Determine the role of the novel DEB-induced putative p53-target genes in the cellular and molecular toxicity of DEB within exposed human lymphocytes. At the completion of these studies, our expectation is that novel DEB-induced direct and/or indirect p53 targets will be identified, and their functions in DEB-exposed cells will be deduced. Collectively, this information is important because it will lead to a better understanding, of not only the toxicity of butadiene at the cellular and molecular level, but also the role of p53 in that process. This knowledge will have an important vertical impact on butadiene risk assessment as well as better means of therapeutic intervention after butadiene exposure and carcinogenesis. PUBLIC HEALTH RELEVANCE: The research proposed in this application is important because it will lead to a better understanding on novel mechanisms of p53 signaling and action as they occur in lymphocytes that have been exposed to the environmental toxicant butadiene, a mutagen and human carcinogen. This knowledge will have an important vertical impact on butadiene risk assessment, as well as on the design of better means of therapeutic intervention after butadiene exposure.